Each year, tens of millions of units of blood are collected worldwide and an equal number of patients receive those units of blood as transfusions. Every unit of collected blood and every patient must be typed for the Rh antigen to ensure a match prior to the transfusion. The ID-Micro Typing System, Inc., disclosed in U.S. Pat. No. 5,338,689 (Ortho Diagnostics, Inc.) provides a simplified method for antigen typing of red blood cells. Other micro typing card systems are also known in the art. For example, a DiaMed AG card system is available from Cressier, Switzerland, known as the DiaMed-ID Micro Typing System. In addition, a Bio Vue card is available from Ortho Diagnostics.
In a conventional blood typing test, drops of typing serum and donor or recipient red blood cells are placed in test tubes and are incubated together. Excess unreacted serum is washed away and a drop of rabbit anti-IgG antibody (Coomb's reagent) is added to the mixture to induce agglutination between cells that may have bound the typing reagent. This test is known as an indirect agglutination test or an indirect Coomb's test. Agglutination is assessed by briefly centrifuging the cells and gently shaking the tubes one by one over a concave mirror and observing the presence of red blood cell agglutinates as the cells return to a suspension. Microwell arrays in microplates may be used in place of test tubes.
In the Micro Typing System, red blood cells are centrifuged in a controlled manner through a dextran-acrylamide gel and Coomb's reagent predispensed in a specially designed microtube. Measured volumes of serum or plasma and/or red blood cells are dispensed into the reaction chamber of the microtube. If necessary, the card is incubated and then centrifuged. Agglutinated red blood cells become trapped in or above the gel and unagglutinated red blood cells travel through the gel particles and form a pellet at the bottom of the microtube.
In a second type of blood group detection system described in WO 9531731 A, a method of detecting a blood group antigen is disclosed. The method comprises adding a sample of red blood cells to a reaction tube which has a lengthwise axis containing a reaction medium consisting of several particles which have immunoglobulin-binding ligands selected from protein A, protein G, protein A/G or a universal kappa light chain binding protein, which ligands are coupled to the surface of the particles, and antibody, optionally a bridging antibody, specific for the antigen coupled to the ligand on the particles. The reaction tube is centrifuged for a time which is sufficient to remove red blood cells which have not attached to the antibody in the form of a pellet in the bottom of the tube. The attachment of the red blood cells, or the lack of attachment of red blood cells is detected and the attachment is correlated with the presence of the antigen.
Each of these methods is designed to detect red blood cell antigens using antibodies which have been produced in eukaryotic cells, either as monoclonal or polyclonal antibodies. These methods cannot be used to detect antibodies which are expressed on the surface of virus particles.
The ability to produce monoclonal antibodies has revolutionized diagnostic and therapeutic medicine. Monoclonal antibodies are typically produced by immortalization of antibody-producing mouse lymphocytes thus ensuring an endless supply of cells which produce mouse antibodies. However, for many human applications, it is desirable to produce human antibodies. For example, it is preferable that antibodies which are administered to humans for either diagnostic or therapeutic purposes are human antibodies since administration of human antibodies to a human circumvents potential immune reactions to the administered antibody, which reactions may negate the purpose for which the antibody was administered.
In addition, there exist certain situations where, for diagnostic purposes, it is essential that human antibodies be used because other animals are unable to make antibodies against the antigen to be detected in the diagnostic method. For example, in order to determine the Rh phenotype of human red blood cells, human sera that contains anti-Rh antibody must be used since no other animal can make an antibody capable of detecting the human Rh antigen.
The production of human antibodies in vitro by immortalizing human B lymphocytes using Epstein Barr virus (EBV)-mediated transformation or cell fusion has been fraught with technical difficulties due to the relatively low efficiency of both EBV-induced transformation and cell fusion when compared with the murine system. To overcome these problems, processes have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57: 191-280). Essentially, a cDNA library is generated from MRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin (Ig) genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab Ig. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human Ig rather than cells which express human Ig.
There are several difficulties associated with the generation of antibodies using bacteriophage. For example, many proteins cannot be purified in a non-denatured state, in that purification procedures necessarily involve solubilization of protein which may render some proteins permanently denatured with concomitant destruction of antigenic sites present thereon. Such proteins thus cannot be bound to a solid phase and therefore cannot be used to pan for phage bearing antibodies which bind to them. An example of such a protein is the human Rh antigen.
To solve the problem, a method was developed wherein intact red blood cells were used as the panning antigen (Siegel et al, 1994, Blood 83: 2334-2344). However, it was discovered that since phage are inherently "sticky" and red blood cells express a multitude of antigens on the cell surface, a sufficient amount of phage which do not express the appropriate antibody on the surface also adhere to the red blood cells, thus rendering the method impractical for isolation of phage which express antibody of desired specificity.
De Kruif et al. (1995, Proc. Natl. Acad Sci. USA 92: 3938-3942) disclose a method of isolating phage encoding antibodies, wherein antibody-expressing phage are incubated with a mixture of antigen-expressing cells and cells which do not express antigen. The antibody-expressing phage bind to the antigen-expressing cells. Following binding with phage, a fluorescently labeled antibody is added specifically to the antigen-expressing cells, which cells are removed from the mixture having antibody-expressing phage bound thereto. The isolation of fluorescently labeled cells is accomplished using the technique of fluorescently-activated cell sorting (FACS), an expensive and time-consuming procedure.
There is a need for a method of isolating recombinant proteins, preferably antibodies, which is rapid and economical, and which will provide a vast array of protein-binding proteins useful for diagnostic and therapeutic applications in humans.
There is also a need for rapid and accurate assays for the typing of red blood cells using recombinant proteins which are expressed on a virus surface. The present invention satisfies these needs.